Vitreous Humor Proteomic Profile in Patients With Vitreoretinal Lymphoma.

Purpose: Vitreoretinal lymphoma is a high-grade malignant non-Hodgkin lymphoma with poor prognosis. The objective of this study was to elucidate the proteome profile of the vitreous in patients with vitreoretinal lymphoma (VRL), aiming to advance understanding of the pathophysiology of VRL. Methods: Comprehensive proteomic analyses of vitreous humor using liquid chromatography with tandem mass spectrometry were performed for 10 patients with VRL, 10 control patients with idiopathic epiretinal membrane or macular hole, and 10 patients with ocular sarcoidosis. Differentially expressed proteins (DEPs) were identified by comparing VRL with controls and sarcoidosis, and functional pathway analysis was performed. Finally, vitreous concentrations of representative DEPs that were significantly upregulated in proteomics study were measured by ELISA using a separate cohort. Results: In total, 1594 proteins were identified in the vitreous humor of VRL, control, and sarcoidosis samples. Also, 282 DEPs were detected in VRL, 249 upregulated and 33 downregulated, compared with controls. Enrichment pathway analysis showed alterations in proteasome-related pathways. Compared to controls and sarcoidosis, 14 DEPs in VRL showed significant upregulation. In the validation study, ELISA confirmed significantly higher vitreous concentrations of PSAT1, YWHAG, and 20S/26S proteasome complex in VRL compared with controls and sarcoidosis. Among the upregulated DEPs, vitreous PITHD1 and NCSTN concentrations correlated positively with vitreous IL-10 concentrations. Conclusions: This study highlights aberrations in protein expression pattern in the vitreous of patients with VRL. The DEPs identified in this study may play pivotal roles in VRL pathogenesis, providing insights to enhance understanding of VRL pathophysiology and contribute to the development of VRL biomarkers.

Arginine mimetic appended peptide-based probes for fluorescence turn-on detection of 14-3-3 proteins.

14-3-3 proteins are adaptor elements in intracellular signaling pathways. Recently, this protein family has been identified as a relevant therapeutic target involved in many human diseases. Therefore, identification of 14-3-3 proteins in biological systems is very important. Two cationic peptide-based probes are reported for the fluorescence detection of 14-3-3 proteins at physiological pH. The design of these probes consists of two symmetric peptidic arms equipped with a guanidiniocarbonyl pyrrole moiety (an arginine mimetic aka GCP), and an environment-sensitive amino-naphthalimide fluorophore as a third arm. These peptide sequences also contain lysine and phenylalanine/tryptophan amino acids for additional charge-charge and hydrophobic interactions. Both probes show high affinity and sensitivity for the 14-3-3 family, as well as good selectivity against other relevant biological proteins and ions.

Combined Short-Term Glucose Starvation and Chemotherapy in 3D Colorectal Cancer Cell Culture Decreases 14-3-3 Family Protein Expression and Phenotypic Response to Therapy.

Short-term glucose starvation prior to chemotherapy has the potential to preferentially weaken cancer cells, making them more likely to succumb to treatment, while protecting normal cells. In this study, we used 3D cell cultures of colorectal cancer and assessed the effects of short-term glucose starvation and chemotherapy compared to treatment of either individually. We evaluated both phenotypic changes and protein expression levels. Our findings indicate that the combined treatment results in more significant phenotypic responses, including decreased cell viability and clonogenicity. These phenotypic responses can be explained by the decreased expression of LDHA and 14-3-3 family proteins, which were found only in the combined treatment groups. This study indicates that short-term glucose starvation has the potential to increase the efficacy of current cancer treatment regimes. Graphical Abstract ᅟ.

Bacterial co-expression of human Tau protein with protein kinase A and 14-3-3 for studies of 14-3-3/phospho-Tau interaction.

Abundant regulatory 14-3-3 proteins have an extremely wide interactome and coordinate multiple cellular events via interaction with specifically phosphorylated partner proteins. Notwithstanding the key role of 14-3-3/phosphotarget interactions in many physiological and pathological processes, they are dramatically underexplored. Here, we focused on the 14-3-3 interaction with human Tau protein associated with the development of several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Among many known phosphorylation sites within Tau, protein kinase A (PKA) phosphorylates several key residues of Tau and induces its tight interaction with 14-3-3 proteins. However, the stoichiometry and mechanism of 14-3-3 interaction with phosphorylated Tau (pTau) are not clearly elucidated. In this work, we describe a simple bacterial co-expression system aimed to facilitate biochemical and structural studies on the 14-3-3/pTau interaction. We show that dual co-expression of human fetal Tau with PKA in Escherichia coli results in multisite Tau phosphorylation including also naturally occurring sites which were not previously considered in the context of 14-3-3 binding. Tau protein co-expressed with PKA displays tight functional interaction with 14-3-3 isoforms of a different type. Upon triple co-expression with 14-3-3 and PKA, Tau protein could be co-purified with 14-3-3 and demonstrates complex which is similar to that formed in vitro between individual 14-3-3 and pTau obtained from dual co-expression. Although used in this study for the specific case of the previously known 14-3-3/pTau interaction, our co-expression system may be useful to study of other selected 14-3-3/phosphotarget interactions and for validations of 14-3-3 complexes identified by other methods.

Alterations in Factors Involved in Differentiation and Barrier Function in the Epithelium in Oral and Genital Lichen Planus.

Lichen planus is a chronic recurrent inflammatory disease affecting both skin and mucosa, mainly in oral and/or genital regions. Keratinocytes go through a well-regulated process of proliferation and differentiation, alterations in which may result in defects in the protective epithelial barrier. Long-term barrier impairment might lead to chronic inflammation. In order to broaden our understanding of the differentiation process in mucosal lichen planus, we mapped the expression of 4 factors known to be involved in differentiation. Biopsies were collected from oral and genital lichen planus lesions and normal controls. Altered expression of all 4 factors in epithelium from lichen planus lesions was found, clearly indicating disturbed epithelial differentiation in lichen planus lesions.

Skin aging caused by intrinsic or extrinsic processes characterized with functional proteomics.

The skin provides protection against environmental stress. However, intrinsic and extrinsic aging causes significant alteration to skin structure and components, which subsequently impairs molecular characteristics and biochemical processes. Here, we have conducted an immunohistological investigation and established the proteome profiles on nude mice skin to verify the specific responses during aging caused by different factors. Our results showed that UVB-elicited aging results in upregulation of proliferating cell nuclear antigen and strong oxidative damage in DNA, whereas chronological aging abolished epidermal cell growth and increased the expression of caspase-14, as well as protein carbonylation. Network analysis indicated that the programmed skin aging activated the ubiquitin system and triggered obvious downregulation of 14-3-3 sigma, which might accelerate the loss of cell growth capacity. On the other hand, UVB stimulation enhanced inflammation and the risk of skin carcinogenesis. Collectively, functional proteomics could provide large-scale investigation of the potent proteins and molecules that play important roles in skin subjected to both intrinsic and extrinsic aging.

Identification of 14-3-3zeta associated protein networks in oral cancer.

Advancements in genomics, proteomics, and bioinformatics have improved our understanding of gene/protein networks involved in intra- and intercellular communication and tumor-host interactions. Using proteomics integrated with bioinformatics, previously we reported overexpression of 14-3-3ζ in premalignant oral lesions and oral squamous cell carcinoma tissues in comparison with normal oral epithelium. 14-3-3ζ emerged as a novel molecular target for therapeutics and a potential prognostic marker in oral squamous cell carcinoma patients. However, the role of 14-3-3ζ in development and progression of oral cancer is not known yet. This study aimed to identify the 14-3-3ζ associated protein networks in oral cancer cell lines using IP-MS/MS and bioinformatics. A total of 287 binding partners of 14-3-3ζ were identified in metastatic (MDA1986) and nonmetastatic (SCC4) oral cancer cell lines including other 14-3-3 isoforms (2%), proteins involved in apoptosis (2%), cytoskeleton (9%), metabolism (16%), and maintenance of redox potential (2%). Our bioinformatics analysis revealed involvement of 14-3-3ζ in protein networks regulating cell cycle, proliferation, apoptosis, cellular trafficking, and endocytosis in oral cancer. In conclusion, our data revealed several novel protein interaction networks involving 14-3-3ζ in oral cancer progression and metastasis.

A five-variable signature predicts radioresistance and prognosis in nasopharyngeal carcinoma patients receiving radical radiotherapy.

Radioresistance poses a major challenge in nasopharyngeal carcinoma (NPC) treatment. Clinical tumor-node-metastasis (TNM) staging has limited accuracy in predicting NPC radioresponse and determining its therapeutic regimens. To construct a risk score model for predicting NPC radioresistance, immunohistochemistry was used to assess the expression of four proteins (14-3-3σ, Maspin, RKIP, and GRP78) in 149 NPC samples with different radiosensitivity. Sequentially, a logistic regression analysis was performed to identify independent predictors of NPC radioresistance and establish a risk score model. As a result, a risk score model, Z = -3.189 - 1.478 (14-3-3σ) - 1.082 (Maspin) - 1.666 (RKIP) + 2.499 (GRP78) + 2.597 (TNM stage), was constructed, and a patient's risk score was estimated by the formula: e (Z)/(e (Z) + 1) × 100, where "e" is the base of natural logarithm. High-risk score was closely associated with NPC radioresistance, and was observed more frequently in the radioresistant patients than that in the radiosensitive patients. The sensitivity, specificity, and accuracy of the risk score model for predicting NPC radioresistance was 88.00, 86.48, and 87.25 %, respectively, which was clearly superior to each individual protein and TNM stage. Furthermore, Kaplan-Meier survival analysis showed that high-risk score correlated with the markedly reduced overall survival (OS) and disease-free survival (DFS) of the patients, and Cox regression analysis showed that the risk score model was an independent predictor for OS and DFS. This study constructs a risk score model for predicting NPC radioresistance and patient survival, and it may serve as a complement to current radioresistance risk stratification approaches.

Upregulation of 14-3-3 eta in chronic liver fluke infection is a potential diagnostic marker of cholangiocarcinoma.

PURPOSE: To discover protein markers in chronic/advanced opisthorchiasis for the early detection of Opisthorchis viverrini (OV)-associated cholangiocarcinoma (CCA). EXPERIMENTAL DESIGN: Liver tissues derived from normal hamsters and those with chronic/advanced opisthorchiasis (n = 5 per group) were subjected to 2DE and LC-MS/MS. Candidate protein expression was confirmed in hamster models and human CCA tissue microarray (TMA) using immunohistochemistry and Western blot. RESULT: Proteomics analysis detected 14-3-3 eta only in infected hamsters, not in uninfected controls. Immunohistochemistry and Western blot analysis confirmed low expression of 14-3-3 eta in normal hamster livers and demonstrated increased expression through time in infected livers. This protein was also observed in parasite organs, especially during the chronic phase of opisthorchiasis. Moreover, increased expression of 14-3-3 eta, relative to normal hamster livers, was observed during the early stage of CCA induced by OV infection and administration of N-nitrosodimethylamine. Immunohistochemical analysis of human TMA revealed that 14-3-3 eta was highly expressed in CCA (84.23%, 187/222 cases) but was not found in hepatocellular carcinoma or healthy liver tissues. CONCLUSIONS AND CLINICAL RELEVANCE: 14-3-3 eta protein has potential as a screening and early diagnostic marker for CCA.

Análise in situ de proteínas diferencialmente expressas em carcinomas epidermoides penianos por espectrometria de massas utilizando Matrix-Assisted Laser Desorption/Ionization (MALDI) / In situ analysis of protein differentially expressed in penile squamous cell carcinoma by Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry

O carcinoma de pênis (CaPe) corresponde a uma doença maligna mutilante do homem. É mais frequente em regiões economicamente desprivilegiadas, como o Norte/Nordeste do Brasil, onde frequentemente é diagnosticado como doença mais avançada. Assim, novos marcadores diagnósticos, prognósticos e preditivos de tratamentos terapêuticos ainda são necessários. Abordagens proteômicas, incluindo o MALDI Imaging, podem contribuir neste sentido. Esta técnica emergente de espectrometria de massas permite a visualização da distribuição espacial de centenas de dados moleculares diretamente da superfície de uma secção tecidual, adquiridos por razão massa/carga (m/z). Neste contexto, nosso principal propósito foi integrar dados de proteômica clássica (gel 2D e Cromatografia Líquida acoplada à Espectrometria de Massas) e de MALDI Imaging, para obter padrões diferenciais de proteínas associados com amostras de Carcinoma Epidermoide Peniano usual (relacionado ou não ao HPV) e espécimes normais, a fim de buscar possíveis biomarcadores da doença. Um total de 45 amostras de CaPe, congeladas, foram inicialmente genotipadas para a presença do HPV. Destas, 60% foram positivas para variantes virais de alto risco. A proteômica clássica (N=24) evidenciou níveis diferenciais de 35 proteínas entre amostras de CaPe e controles, e 29 entre CaPe HPV positivo versus negativo (P<0,05; ANOVA). Redes de interações demonstraram que estes perfis proteicos interagiam com clusters de proteínas relacionadas com a carcinogênese e progressão tumoral. Entre eles, se destacaram aqueles formados por proteínas antioxidantes e de adesão celular, presentes em níveis elevados em tumores HPV negativos. A partir dos interactomas, quatro alvos proteicos foram selecionados para a análise in situ por imageamento: Calreticulina, 14-3-3 sigma, Serpina B5 e Glutationa-s-transferase. A aquisição de dados do MALDI Imaging foi conduzida após a digestão in situ pela tripsina, usando uma resolução de 200 µm e faixa de 700-3500 m/z para peptídeos (N=31). Os dados de identificação do gel 2D foram então integrados aos do imageamento. A identidade proteica dos filtros foi confirmada, in silico, por meio da presença de peptídeos teóricos co-localizados com o peptídeo experimental alvo nas secções de CaPe. Não houve associação significativa entre os parâmetros clinicopatológicos e as intensidades de sinal dos alvos (P>0,05, U de Mann-Whitney). Análises não supervisionadas, realizadas a partir dos dados do MALDI Imaging, evidenciaram mapas de segmentação que coindiciram com as regiões tumorais e margens adjacentes livres de neoplasia. Entre os principais valores de m/z diferenciadores estava o pico 1413 ± 2,5 Da, abundante nas regiões tumorais, e correlacionado ao peptídeo experimental m/z 1410,86 referente à proteína Calreticulina (CRT), o. Análises estatísticas (PCA e Curva ROC) indicaram este valor de m/z como potencial biomarcador da doença. Por conseguinte, a CRT foi selecionada para a etapa de validação por imunoistoquímica em tecidos parafinados de CaPe (N=158). Níveis elevados de imunoreatividade da CRT foram associados com piores tempo de sobrevida global (Razão de Risco 2,3; IC-1,46-3,96; P<0,001) e câncer específica (Razão de Risco 4,37; IC-1,66-11,51; P=0,002) nos casos de CaPe. A presença de metástase em linfonodos foi considerado um fator prognóstico independente para o risco de morte pelo câncer (Razão de Risco ­ 14,18; CI-3,29-61,12; P <0,001). A imunoreatividade da CRT também foi capaz de predizer a presença de metástase em linfonodos (Chance de Risco: 1,006; IC- 1,0001-1,0012; p=0,044). Estes dados, em conjunto, sugerem que a CRT pode ser um potencial biomarcador prognóstico do CaPe. A estratégia de integração da proteômica clássica com o MALDI Imaging, mostrou-se uma ferramenta útil na busca de novos biomarcadores para o CaPe. Além disto, o trabalho adicionou uma visão analítica à histopatologia clássica, o que deverá inserir as técnicas utilizadas neste projeto em estudos de Anatomia Patológica, tanto em nossa instituição, quanto no contexto global.

Penile cancer (PeCa) corresponds to a mutilating malignant disease in men. It is more frequent in underprivileged socioeconomic regions (e.g., Noth, North-East of Brazil), where it is frequently diagnosed in advanced stages. Thus, new markers are still needed for early diagnosis, prognosis and prediction of therapy. Proteomic approaches, including MALDI Imaging, could assist in this effort. This emerging spatially resolved mass spectrometric technique can obtain topographical distribution of hundreds of molecules directly from the tissue section surface, mensured by mass/charge ratio (m/z). In this context, our mainly propose was to integrate classic proteomic data (2D gel and Liquid Chromatograph coupled with Mass Spectrometry) with MALDI Imaging to obtain diferential patterns of protein associated with Usual Squamous Cell Penile Carcinoma (HPV related or not) and normal specimens, to look for possible biomarkers of the disease. A total of 45 fresh-frozen PeCa samples were initially searched for HPV genotype, 60% of which were positive for high-risk HPV. Classic proteomics (N=24) demonstrated diferential levels of 35 proteins comparing PeCa and control samples, and 29 comparing HPV-positive versus HPV-negative PeCa samples (P<0.05; ANOVA). Protein networks showed that these protein profiles interact with clusters of proteins related with tumorigenesis and tumor progression processes. Among them, antioxidant and cell adhesion proteins play a critical role in HPV negative penile tumors. Based on interactome data, four protein targets were selected for in situ analyses by imaging: Calreticulin, 14-3-3 protein sigma, Serpin B5 and Glutatione-s-transferase. MALDI Imaging data acquisition of peptides was conducted after in situ trypsin digestion using a lateral resolution of 200 µm, covering the range 700- 3500 m/z (N=31). After that, 2D gel based proteomic data was integrated with Imaging data. The filter protein identities were confirmed in silico by the co-localization of theoretical triptic peptides with the experimental peptides in PeCa sections. There was no significant association between the clinical and pathological parameters and the target signal intensities (P>0.05; U de Mann-Whitney). An unsupervised clustering analysis based on MALDI Imaging data reveled segmentation maps that coincide with histological annotation for tumor and adjacent non-neoplasic regions. Among the mainly differentiating m/z values there was 1413 ± 2.5 Da. This peak was especially co-localized with tumoral regions and correlated with Calreticulin (CRT) experimental peptide (m/z 1410,86). Statistical analysis (PCA and ROC Curves) indicated this m/z value as a potencial biomarker of the disease. For this reason, CRT was selected for validation by immunohistochemistry performed on paraffin-embedded PeCa tissues (N=158). As result, CRT hiperexpression in PeCa tissue increased the risk of unfavorable overall survival (Relative Risk ­ 2.3; CI-1.46-3.96; P<0.001) and cancer specific survival (Relative Risk ­ 4.37; CI-1.66-11.51; P=0.002) in these patients. Lymph node metastasis represented an independent prognostic risk factor for death related to cancer in our patients (Relative risk ­ 14.18; CI-3.29-61.12; P <0.001). CRT immunoreactivity was also capable to predict the presence of lymph node metastases (Risk Chance ­ 1,006; CI-1.0001-1.00123; P =0.044). Taken together, our results sugest that CRT may represent a prognostic biomarker of PeCa. The strategy of integrated classic proteomic and MALDI Imaging revealed as usefull tool to search for news biomarkers of the disease. Futhermore, this work added an analytical perspective to the classical histopathology, allowing to include the techniques used in this project in future morphological studies, both in our institution and in the global context.

Keratin 17 is co-expressed with 14-3-3 sigma in oral carcinoma in situ and squamous cell carcinoma and modulates cell proliferation and size but not cell migration.

Expression of keratin (K) 13 is replaced with that of K17 when squamous cells of the oral mucosa transform from normal and dysplastic epithelia to carcinoma in situ (CIS) and squamous cell carcinoma (SCC). Since 14-3-3 sigma is functionally associated with K17, we examined possible relationships between expression of K17 and 14-3-3 sigma in oral CIS and SCC tissues by immunohistochemistry. We furthermore examined whether or not K17 expression or knockdown by small interfering RNA (siRNA) modulates the behavior of SCC cells in culture in terms of cell proliferation and migration. In tissue specimens of oral SCC and CIS, the pattern of cytoplasmic expression of 14-3-3 sigma and K17 was similar but neither was expressed in normal or dysplastic epithelia. Both proteins were demonstrated in the cytoplasm of control oral SCC ZK-1 cells, but expression of 14-3-3 sigma changed from cytoplasmic to nuclear upon knockdown of K17. In carcinoma cells, therefore, cytoplasmic localization of 14-3-3 sigma seems to accompany expression of K17. In K17-knockdown cells, proliferation was significantly suppressed at 4 days after seeding. In addition, the cell size of K17-knockdown cells was significantly smaller than that of control cells; as a result of which in the migration experiments, we found delayed closure of scratch wounds but migration as such was not affected. We conclude that K17 expression promotes SCC cell growth and cell size but does not affect cell migration. K17 expression is accompanied by cytoplasmic expression of 14-3-3 sigma, indicative of their functional relationship.

Immunocytochemical staining for stratifin and OCIAD2 in bronchial washing specimens increases sensitivity for diagnosis of lung cancer.

OBJECTIVE: Brushing or washing cytology taken at bronchoscopy is a standard diagnostic procedure for lung cancer. The present study evaluated the sensitivity of immunocytochemical diagnosis of lung cancer using bronchial washing materials. METHODS: We collected bronchial washing samples taken at bronchoscopy between July 2012 and July 2013 at Tsukuba University Hospital and studied 106 cases that were finally diagnosed as lung cancer. We collected exfoliated cells using a thin-layer advanced cytology assay system (TACAS(™)) and performed cytological diagnosis using Papanicolaou staining. As controls, we randomly selected 30 tumour-negative cases from among samples collected during the same period. Using these materials, we also examined the expression of stratifin (14-3-3 sigma) (n = 92) and OCIAD2 ovarian immunoreactive antigen domain 2) (n = 106) by immunocytochemistry, as these are considered to be broad spectrum immune markers for lung adenocarcinoma including early invasive lung adenocarcinoma. RESULTS: Using Papanicolaou staining, 52 out of 106 lung cancers (49.1%) were diagnosed as positive. However, positivity was increased to 63.0% by immunocytochemistry using anti-stratifin or anti-OCIAD2 antibodies. Biopsies were taken in 103/106 cases and cancer was diagnosed in 60/103, (58.3%). The sensitivity of stratifin or OCIAD2 was significantly higher than that of Papanicolaou staining (P = 0.027), but immunocytochemistry detected false-positive cells in 3/30 cases (10%) for stratifin and 2/30 cases (7%) for OCIAD2. CONCLUSION: Immunocytochemical staining for stratifin and OCIAD2 improved diagnostic sensitivity for lung cancers but diagnostic specificity was lower than that for cytology alone. The immunostains carried up to a 10% risk of a false-positive result and therefore positive staining must be confirmed by morphological evidence of malignancy.

Prognostic significance of YWHAZ expression in localized prostate cancer.

BACKGROUND: Prostate cancer (PCa) patients are often over-treated because of the lack of biomarkers needed to distinguish the lethal from the indolent form of PCa. YWHAZ was recently identified as a potential therapeutic target in castration-resistant PCa (CRPC). Therefore, this study focused on determining the prognostic significance of YWHAZ in localized PCa. METHODS: YWHAZ expression was assessed by immunohistochemistry on formalin-fixed paraffin-embedded tissue from 213 men who underwent radical prostatectomy. Kaplan-Meier analysis and Cox proportional-hazards models were used to assess the prognostic value of YWHAZ intensity. RESULTS: High YWHAZ expression was strongly associated with high Gleason score at the time of diagnosis (P < 0.001) and PSA relapse (P = 0.001). Importantly, patients with high expression of YWHAZ had a higher risk of CRPC development (P = 0.002) and reduced survival time (P = 0.002). CONCLUSIONS: Our findings indicate that YWHAZ could serve as a promising prognostic biomarker in localized PCa to predict poor prognosis and to identify a subgroup of tumors, which might benefit from earlier adjuvant or YWHAZ-targeted therapy.

Identification of proteins associated with lymph node metastasis of gastric cancer.

PURPOSE: The aim of this study was to identify proteins associated with gastric cancer lymph node metastasis and explore the clinicopathological significance of these proteins. METHODS: Gastric cancer tissues were obtained from 24 patients with high or low lymph node metastatic potential. Total cellular proteins were separated by two-dimensional gel electrophoresis (2-DE), analyzed by MALDI/TOF-TOF MS, and identified by a database search. Expression of 14-3-3ß and profilin-1 was then immunohistochemically verified in paraffin-embedded gastric cancer tissues from 128 patients and analyzed by multivariate logistic regression models, Kaplan-Meier curves, and Cox proportional hazard models. RESULTS: A total of 26 differentially expressed proteins were identified, 20 of which were overexpressed and 6 of which were underexpressed. 14-3-3ß and profilin-1 were upregulated in gastric cancer tissues with and without lymph node metastasis, respectively. Expression of 14-3-3ß protein was associated, but profilin-1 expression was inversely associated with gastric cancer lymph node metastasis. Multivariate analysis showed that overexpression of 14-3-3ß and reduced expression of profilin-1 were independent risk factors for gastric cancer lymph node metastasis, while 14-3-3ß overexpression was an independent prognostic factor for gastric cancer patients. CONCLUSIONS: The current study identified 26 differentially expressed proteins. Further studies showed that both 14-3-3ß and profilin-1 protein may be useful biomarkers for prediction of gastric cancer lymph node metastasis and that expression of 14-3-3ß was a prognostic marker for gastric cancer patients.

Expression profiles for 14-3-3 zeta and CCL20 in pancreatic cancer and chronic pancreatitis.

BACKGROUND: Pancreatic cancer (PCA) has a dismal prognosis because it is often diagnosed at an advanced stage. The overall survival rate is <5% after five years. Chronic pancreatitis (CP) is one of the most important risk factors for PCA. A major difficulty is to distinguish between CP and PCA at both clinical and morphologic level. The aim of this study was to assess the impact of the expression profiles for 14-3-3 zeta and CCL20 to histologically discriminate between PCA and CP. METHODS: In PCA (n=138) and CP (n=36) tissue samples, the expression of 14-3-3 zeta and CCL20 was examined by immunohistochemistry. Associations between expression profiles of 14-3-3 zeta and CCL20 expression in PCA, CP as well as MANT (matched adjacent normal tissue) (n=138) and clinicopathologic variables were analyzed. RESULTS: The expression of CCL20 and 14-3-3 zeta was significantly higher in PCA than in CP and MANT. For CP compared to MANT, no significant differences were observed for expression profiles of both 14-3-3 zeta and CCL20. CONCLUSION: CCL20 and 14-3-3 zeta are molecules that play a putative role during tumorgenesis in pancreas, and may therefore be new parameters for histological diagnosis and discrimination between PCA and CP.

Evaluation of fluorescence in-situ hybridization in monomorphic endometrial stromal neoplasms and their histological mimics: a review of 49 cases.

AIMS: To perform a population-based review of monomorphic endometrial stromal tumours and their histological mimics presenting over a 20-year period, including an evaluation of fluorescence in-situ hybridization (FISH) for the JAZF1 and YWHAE breakaparts. METHODS AND RESULTS: Forty-nine tumours were examined, comprising 13 histological mimics and 36 endometrial stromal tumours [six stromal nodules (ESNs), 25 low-grade stromal sarcomas (ESSs), and five monomorphic undifferentiated sarcomas (mUESs)]. Nine ESSs showed variant histological patterns, including smooth muscle, sex cord-like/glandular, fibrous or rhabdoid differentiation. Three ESSs were initially misclassified as benign uterine lesions, and, conversely, three benign mimics were originally reported as ESSs. One mUES showed a prominent pseudopapillary pattern. Fluorescence in-situ hybridization demonstrated JAZF1 breakaparts in five of six ESNs and 16 of 25 ESSs; however, only three of nine ESS variants were positive. YWHAE breakaparts were present in four of five mUESs. Analysis of a subsequent metastasis in the YWHAE breakapart-negative mUES demonstrated a YWHAE deletion. None of the histological mimics was positive in FISH analysis. Diffuse cyclin D1 expression was restricted to mUESs in this series. CONCLUSIONS: Endometrial stromal neoplasms continue to present diagnostic difficulty. Fluorescence in-situ hybridization analysis is helpful in distinguishing stromal tumours from their histological mimics and in distinguishing ESS from mUES.

Expression of 14-3-3 sigma, cyclin-dependent kinases 2 and 4, p16, and Epstein-Barr nuclear antigen 1 in nasopharyngeal carcinoma.

OBJECTIVE: The protein 14-3-3 sigma plays a role in cell cycle arrest by sequestering cyclin-dependent kinase 1 cyclin B1 complexes, as well as cyclin-dependent kinases 2 and 4, hence its definition as a cyclin-dependent kinase inhibitor. However, the nature of the interaction between these biological markers in nasopharyngeal carcinoma is unknown. This study aimed to investigate whether altered expression of these markers contributes to nasopharyngeal carcinogenesis. METHODS: The study population consisted of 30 nasopharyngeal carcinoma patients and 10 patients without nasopharyngeal carcinoma. The nasopharyngeal carcinoma cell lines TW02, TW04 and Hone-1 were also assessed. We analysed levels of messenger RNA and protein for the p16 gene and the 14-3-3 sigma, Epstein-Barr nuclear antigen 1, and cyclin-dependent kinase 2 and 4 proteins, in nasopharyngeal carcinoma tissue specimens and cell lines and in normal nasopharyngeal tissue. RESULTS: Protein and messenger RNA levels for cyclin-dependent kinase 2 and Epstein-Barr nuclear antigen 1 were significantly higher in nasopharyngeal carcinoma compared with normal tissue, while levels of cyclin-dependent kinase 4 generally were not; results for 14-3-3 sigma varied. Nasopharyngeal carcinoma patients had diminished p16 gene expression, compared with normal tissue. CONCLUSION: Levels of cyclin-dependent kinase 2 and Epstein-Barr nuclear antigen 1 were significantly higher in nasopharyngeal carcinoma than in normal tissue, while p16 gene expression was diminished. These three proteins may contribute to nasopharyngeal carcinogenesis.

The usefulness of S100P, mesothelin, fascin, prostate stem cell antigen, and 14-3-3 sigma in diagnosing pancreatic adenocarcinoma in cytological specimens obtained by endoscopic ultrasound guided fine-needle aspiration.

Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) of the pancreas is an efficient and minimally invasive procedure for the diagnosis and staging of pancreatic adenocarcinoma. Because of some limitations of EUS-FNA in diagnosis of well-differentiated or early stage cancers, the purpose of this study is to assess the added benefit of immunohistochemistry. We studied five proteins overexpressed in pancreatic adenocarcinoma, namely, prostate stem cell antigen, fascin, 14-3-3 sigma, mesothelin and S100P utilizing immunohistochemistry on paraffin sections from cellblocks obtained by EUS-FNA. Sixty-two cases of EUS-FNA of the pancreas that had follow-up histological and/or clinical diagnosis and sufficient material in cell blocks were included. Using histological diagnosis and/or clinical outcome as the reference standard, EUS-FNA shows the highest sensitivity (95%) and specificity (91%) and is superior to any marker in this study. Among five antibodies, S100P reveals the best diagnostic characters showing 90% of sensitivity and 67% of specificity. Fascin shows high specificity (92%) but low sensitivity (38%). Mesothelin has a moderate sensitivity (74%) and low specificity (33%), PSCA and 14-3-3 show high sensitivity but zero specificity. S100P and mesothelin were useful in nine indeterminate cases. S100P correctly predicted six of seven cancers and one of one without cancer and mesothelin correctly diagnosed five of seven cancers and one of two noncancers in this group. EUS-FNA cytomorphology is superior to any of the immunohistochemical markers used in this study. Use of S100P and mesothelin in cytologically borderline cases can increase the diagnostic accuracy in this group.

[Effect of acupuncture intervention on 14-3-3 expression in cerebral cortex of hypoxic-ischemic brain damage rats].

OBJECTIVE: To observe the effect of acupuncture therapy on 14-3-3, Bcl-2 and Bax expression levels in the cerebral cortex in neonatal rats with hypoxic-ischemic brain damage(HIBD). METHODS: Timed pregnant Sprague-Dawley rat dams were delivered either vaginally (normal group), or by C-section (sham-operation group) or by C-section with 5 min of global anoxia (anoxia group), with 8 rats in each group. The rat pups of the anoxia group were randomly divided into model group and acupuncture group (n =8). Acupuncture stimulation of "Naosanzhen" "Niesanzhen" and "Zhisanzhen" acupoints was given begin- ning from the 14th day after birth, once daily for 7 consecutive days. All rat pups were killed by decapitation on day 21 after birth, and then 14-3-3, Bcl-2 and Bax immunoactivity (expression) in the cerebral cortex were detected by immunohistochemistry. RESULTS: In comparison with the normal group, the expression level of cerebral cortical 14-3-3 was significantly decreased, and that of Bax remarkably increased in the model group (P<0. 01, P<0. 05). Compared to the model group, cortical 14-3-3 and Bcl-2 expression levels were markedly up-regulated in the acupuncture group (P<0.01, P<0.05). Compared to the normal group, cortical 14-3-3 expression level was obviously lower, but Bax expression level significantly higher in the sham-operation group (P<0. 05, P<0. 01). No significant differences were found between the model and normal groups in the expression levels of Bcl-2, and between the acupuncture and model groups in the expression levels of Bax (P>0. 05). CONCLUSION: Acupuncture intervention can increase the expression of 14-3-3 and Bcl-2 in the cerebral cortex in HIBD rats.

Dual phosphorylation of Btk by Akt/protein kinase b provides docking for 14-3-3ζ, regulates shuttling, and attenuates both tonic and induced signaling in B cells.

Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in the Btk gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.